Fasudil regulates T cell responses through polarization of BV-2 cells in mice experimental autoimmune encephalomyelitis
Abstract
Chan CHEN1, Yan-hua LI2, Qiong ZHANG1, Jie-zhong YU2, Yong-fei ZHAO1, Cun-gen MA2, 3, *, Bao-guo XIAO1, *
1Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040, China; 2Institute of Brain Science, Department of Neurology, Medical School, Shanxi Datong University, Datong 037009, China; 3Department of Encephalopathy and National Major Clinical Department of Ministry of Health, Third Hospital, Shanxi University of Traditional Chinese Medicine, Taiyuan 030006, China
Aim: Fasudil, a selective Rho kinase (ROCK) inhibitor, has been shown to alleviate the severity of experimental autoimmune encephalomyelitis (EAE) via attenuating demyelination and neuroinflammation. The aim of this study was to investigate the effects of fasudil on interactions between macrophages/microglia and T cells in a mice EAE model.
Methods: Mouse BV-2 microglia were treated with IFN-γ and fasudil. Cell viability was detected with MTT assay. BV-2 microglia polarization was analyzed using flow cytometry. Cytokines and other proteins were detected with ELISA and Western blotting, respectively. Mice were immunized with MOG35–55 to induce EAE, and then treated with fasudil (40 mg/kg, ip) every other day from d 3 to d 27 pi. Encephalomyelitic T cells were prepared from the spleen of mice immunized with MOG35–55 on d 9 pi.
Results: Treatment of mouse BV-2 microglia with fasudil (15 μg/mL) induced significant phenotype polarization and functional plasticity, shifting M1 to M2 polarization. When co-cultured with the encephalomyelitic T cells, fasudil-treated BV-2 microglia significantly inhibited the proliferation of antigen-reactive T cells, and down-regulated IL-17-expressing CD4+ T cells and IL-17 production. Furthermore, fasudil-treated BV-2 microglia significantly up-regulated CD4+CD25high and CD4+IL-10+ regulatory T cells (Tregs) and IL-10 production, suggesting that the encephalomyelitic T cells had converted to Tregs. In EAE mice, fasudil administration significantly decreased both CD11b+iNOS+ and CD11b+TNF-α+ M1 microglia, and increased CD11b+IL-10+ M2 microglia.
Conclusion: Fasudil polarizes BV-2 microglia into M2 cells, which convert the encephalomyelitic T cells into Tregs in the mice EAE model.
Keywords: fasudil; multiple sclerosis; experimental autoimmune encephalomyelitis; neuroinflammation; microglia; T cell; cell polarization; cytokine
This work was supported by grants from the National Natural Science Foundation of China (No 81070957, 81371414, and 81070956).
* To whom correspondence should be addressed.
E-mail bgxiao@shmu.edu.cn (Bao-guo XIAO); macungen2001@163.com (Cun-gen MA)
Received 2014-01-13 Accepted 2014-04-22
Keywords:
1Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200040, China; 2Institute of Brain Science, Department of Neurology, Medical School, Shanxi Datong University, Datong 037009, China; 3Department of Encephalopathy and National Major Clinical Department of Ministry of Health, Third Hospital, Shanxi University of Traditional Chinese Medicine, Taiyuan 030006, China
Aim: Fasudil, a selective Rho kinase (ROCK) inhibitor, has been shown to alleviate the severity of experimental autoimmune encephalomyelitis (EAE) via attenuating demyelination and neuroinflammation. The aim of this study was to investigate the effects of fasudil on interactions between macrophages/microglia and T cells in a mice EAE model.
Methods: Mouse BV-2 microglia were treated with IFN-γ and fasudil. Cell viability was detected with MTT assay. BV-2 microglia polarization was analyzed using flow cytometry. Cytokines and other proteins were detected with ELISA and Western blotting, respectively. Mice were immunized with MOG35–55 to induce EAE, and then treated with fasudil (40 mg/kg, ip) every other day from d 3 to d 27 pi. Encephalomyelitic T cells were prepared from the spleen of mice immunized with MOG35–55 on d 9 pi.
Results: Treatment of mouse BV-2 microglia with fasudil (15 μg/mL) induced significant phenotype polarization and functional plasticity, shifting M1 to M2 polarization. When co-cultured with the encephalomyelitic T cells, fasudil-treated BV-2 microglia significantly inhibited the proliferation of antigen-reactive T cells, and down-regulated IL-17-expressing CD4+ T cells and IL-17 production. Furthermore, fasudil-treated BV-2 microglia significantly up-regulated CD4+CD25high and CD4+IL-10+ regulatory T cells (Tregs) and IL-10 production, suggesting that the encephalomyelitic T cells had converted to Tregs. In EAE mice, fasudil administration significantly decreased both CD11b+iNOS+ and CD11b+TNF-α+ M1 microglia, and increased CD11b+IL-10+ M2 microglia.
Conclusion: Fasudil polarizes BV-2 microglia into M2 cells, which convert the encephalomyelitic T cells into Tregs in the mice EAE model.
Keywords: fasudil; multiple sclerosis; experimental autoimmune encephalomyelitis; neuroinflammation; microglia; T cell; cell polarization; cytokine
This work was supported by grants from the National Natural Science Foundation of China (No 81070957, 81371414, and 81070956).
* To whom correspondence should be addressed.
E-mail bgxiao@shmu.edu.cn (Bao-guo XIAO); macungen2001@163.com (Cun-gen MA)
Received 2014-01-13 Accepted 2014-04-22